RESUMEN
An activating mutation of Fms-like tyrosine kinase 3 (FLT3) is the most frequent genetic alteration associated with poor prognosis in acute myeloid leukemia (AML). Although many FLT3 inhibitors have been clinically developed, no first-generation inhibitors have demonstrated clinical efficacy by monotherapy, due to poor pharmacokinetics or unfavorable safety profiles possibly associated with low selectivity against FLT3 kinase. Recently, a selective FLT3 inhibitor, quizartinib, demonstrated favorable outcomes in clinical studies. However, several resistant mutations emerged during the disease progression. To overcome these problems, we developed a novel FLT3 inhibitor, FF-10101, designed to possess selective and irreversible FLT3 inhibition. The co-crystal structure of FLT3 protein bound to FF-10101 revealed the formation of a covalent bond between FF-10101 and the cysteine residue at 695 of FLT3. The unique binding brought high selectivity and inhibitory activity against FLT3 kinase. FF-10101 showed potent growth inhibitory effects on human AML cell lines harboring FLT3 internal tandem duplication (FLT3-ITD), MOLM-13, MOLM-14, and MV4-11, and all tested types of mutant FLT3-expressing 32D cells including quizartinib-resistant mutations at D835, Y842, and F691 residues in the FLT3 kinase domain. In mouse subcutaneous implantation models, orally administered FF-10101 showed significant growth inhibitory effect on FLT3-ITD-D835Y- and FLT3-ITD-F691L-expressing 32D cells. Furthermore, FF-10101 potently inhibited growth of primary AML cells harboring either FLT3-ITD or FLT3-D835 mutation in vitro and in vivo. These results indicate that FF-10101 is a promising agent for the treatment of patients with AML with FLT3 mutations, including the activation loop mutations clinically identified as quizartinib-resistant mutations.
Asunto(s)
Amidas/uso terapéutico , Antineoplásicos/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Mutación , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/uso terapéutico , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/genética , Amidas/farmacocinética , Amidas/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Línea Celular Tumoral , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Tirosina Quinasa 3 Similar a fms/químicaRESUMEN
Ceramide kinase (CerK) catalyzes the conversion of ceramide to ceramide 1-phosphate (C1P). We previously revealed that CerK is involved in the activation of mast cells. In this study, we performed an advanced investigation into the role of CerK on the activation of mast cells using CERK-/- mice. Although CERK-/- mice were less prone to exhibiting a passive cutaneous anaphylactic shock (PCA)-reaction compared to wild type (WT) mice, the differences were not significant. In bone marrow-derived mast cells (BMMC) activated by cross-linking antigen (Ag)/IgE, not high, but low concentrations of Ag had a reduced effect on degranulation in BMMC from CERK-/- mice compared to effects on BMMC from WT mice. Similarly, when the BMMCs were activated with calcium ionophore to focus on the downstream signaling of Ca(2+)-elevation, only a low concentration of ionophore had a reduced effect on degranulation in the BMMC from CERK-/- mice compared to the effect on BMMC from WT mice. Furthermore, the CerK inhibitor K1 reduced the differences in degranulation observed between the BMMC from CERK-/- and WT mice in a dose-dependent manner, demonstrating a contribution for CerK and its product C1P in degranulation. Although CerK is not essential for activation of mast cells, especially a potent and acute activation such as a PCA reaction, CerK might act as an modulator for mild and chronic activation of mast cells, thus increasing sensitivity to cytoplasmic Ca(2+).
Asunto(s)
Calcio/metabolismo , Mastocitos/citología , Mastocitos/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Células de la Médula Ósea/citología , Degranulación de la Célula/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/deficienciaRESUMEN
Ceramide kinase (CerK) has important roles in leukocyte functions, including the role in degranulation of mast cells and the phagocytosis of polymorphonuclear leukocytes, so its expression levels should be strictly regulated. Here, we report that the mRNA expression and enzyme activity of CerK were decreased during macrophage-like cell differentiation of the leukemia cell line HL-60, yet neither was altered during granulocytic differentiation of the same cells. Our findings demonstrate that HL-60 cells are useful for studying CerK functions in leukocyte differentiation, and they also suggest that CerK might have an important role in such differentiation.
Asunto(s)
Diferenciación Celular , Leucemia/enzimología , Leucemia/patología , Macrófagos/citología , Macrófagos/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Células HL-60 , Humanos , Macrófagos/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Tretinoina/farmacologíaRESUMEN
Ceramide kinase (CERK) is an enzyme that phosphorylates ceramide to produce ceramide 1-phosphate. Recently, evidence has emerged that CERK has a role in inflammatory signaling of immune cells. Since obesity is accompanied by chronic, low-grade inflammation, we examined whether CERK might be involved using CERK-null mice. We determined that CERK deficiency suppresses diet-induced increases in body weight, and improves glucose intolerance. Furthermore, we demonstrated that CERK deficiency attenuates MCP-1/CCR2 signaling in macrophages infiltrating the adipose tissue, resulting in the suppression of inflammation in adipocytes, which might otherwise lead to obesity and diabetes.